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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 May - 01 Jul 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Appearance: white, waxy solid
- Source and lot/batch No.of test material: 605803
- Expiration date of the lot/batch: 31 Dec 2016
- Purity: 98.4%
- Purity test date: 25 Nov 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Substance is hydrolytically active
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Because the test substance is not stable in aqueous solution, the substance was melted and applied to watch glasses. After cooling and weighing, the watch glasses were added directly to test vessels containing 200 mL tap water purified by reverse osmosis.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.9 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
Not reported
Test temperature:
18 - 21 °C
pH:
Start, 7.7 - 7.8 before addition of sludge
7.4 - 8.4 after 3-hr exposure period
Nominal and measured concentrations:
Nominal concentrations only: 10 mg/L, 32 mg/L, 100 mg/L, 320 mg/L, and 1000 mg/L during exposure period.
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass bottles/vessels.
- Type (delete if not applicable): Open during exposure. Respiration measured in closed cell with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany) multichannel measuring and controlling system.
- Material, size, headspace, fill volume: 200 mL purified water plus test substance, 16 mL synthetic sewage, and 250 mL sludge suspension plus sufficient RO water to bring total volume to 500 mL.
- Aeration: Continuous aeration and stirring.
- No. of vessels per concentration (replicates) 5
- No. of vessels per control (replicates): 6
- Biomass loading rate: 1.5 g/L suspended solids in the final test mixture.
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO)

OTHER TEST CONDITIONS
- Adjustment of pH: none.
- Light intensity: not specified.

EFFECT PARAMETERS MEASURED : Dissolved oxygen concentration.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric series (log 0.5)
- Range finding study: Yes
- Test concentrations: 10, 100 and 1000 mg/L, with and without nitrification inhibitor
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 30% effect at highest level tested
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
139 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Based on results of the preliminary test, MTDID 15670 is inhibitory to nitrification. Total respiration was measured in the final test because there was no evidence of bi-phasic or distorted dose-response.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 12 mg/L The EC50 was within the 2 to 25 mg/L range considered acceptable for the test.
Reported statistics and error estimates:
Estimation of the EC50 value for the reference substance was based on Weibull analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.

Calculation of EC10 values for MTDID 15670 were based on the 3-param. normal Cumulative Distribution Function using a non-linear regression without weighting. An EC50 for MTDID 15670 could not be calculated because effects were below 50%.

The NOEC was determined using Williams Multiple Sequential t-test Procedure (α=0.05, onesided, smaller) to examine statistical significance of respiration rate at each each test substance level v. control.

Sample

Conc. or loading rate

(mg/L)

Respiration rate

(mg O2)/ (L∙h)

Respiration rate

(mg O2)/ (g∙h)

% Inhibition relative to control mean

Blank controls

C1

0

46.54

31.03

C2

0

40.75

27.17

C3

0

37.04

24.69

C4

0

47.14

31.43

C5

0

42.71

28.47

C6

0

49.45

32.96

Mean

43.94

29.29

SD

4.62

3.08

CV (%)

11

11

Reference sub.¹

ref 1

2.0

38.97

25.98

2

ref 2

4.0

27.37

18.24

31

ref 3

8.0

26.61

17.74

33

ref 4

16

16.33

10.89

59

Test sub.

T1a

10

40.03

26.69

9

T1b

10

38.98

25.99

11

T1c

10

34.99

23.33

20

T1d

10

39.95

26.63

9

T1e

10

43.08

28.72

2

Mean T1

39.41

26.27

10

T2a

32

40.22

26.81

8

T2b

32

39.69

26.46

10

T2c

32

40.16

26.77

9

T2d

32

40.52

27.01

8

T2e

32

43.14

28.76

2

Mean T2

40.74

27.16

7

T3a

100

39.30

26.20

11

T3b

100

46.52

31.01

-6

T3c

100

45.57

30.38

-4

T3d

100

39.01

26.01

11

T3e

100

40.19

26.80

9

Mean T3

42.12

28.08

4

T4a

320

41.54

27.69

5

T4b

320

33.31

22.21

24

T4c²

320

#

#

#

T4d

320

28.88

19.26

34

T4e

320

29.58

19.72

33

Mean T4

 

33.33

22.22

24³

 

 

 

 

 

T5a

1000

26.32

17.54

40

T5b

1000

36.51

24.34

17

T5c

1000

29.96

19.97

32

T5d

1000

24.57

16.38

44

T5e

1000

35.65

23.77

19

Mean T5

 

30.60

20.40

30³

1, reference substance was 3,5-dichlorophenol

2, respiration rate could not be calculated due to non-linearity in oxygen curve

3, Mean significantly different than control

Validity criteria fulfilled:
yes
Remarks:
Respiration rate in blank controls >20 mg O2/g∙h (29 mg O2/g∙h). Coefficient of variation of control respiration <30% (11%). EC50 for 3,5-DCP in range 2-25 mg/L (12 mg/L)
Conclusions:
MTDID 15670 is not inhibitory to sludge at 100 mg/L. 50% effect was not attained up to 1000 mg/L (OECD209)
Executive summary:

The toxicity of MTDID 15670 to activated sludge was assessed according to the OECD 209 guideline. The test substance was melted, applied to watch glasses, and added directly to test vessels owing to difficulty of working with aqueous solutions. Some effect on nitrification was noted in a preliminary test, however overall effects were small. In the definitive test, 50% effect was not attained at 1000 mg/L. The NOEC was 100 mg/L, and the calculated EC10 was 139 mg/L.

The study was performed in accordance with internationally-accepted test guidelines under GLP. The study is deemed reliable without restriction and suitable for use in Risk Assessment, Classification & Labeling, and PBT Analysis.

Description of key information

MTDID 15670 is of limited direct toxicity to microorganism (EC10, 139 mg/L, 50% effect not attained at 1000 mg/L maximum loading rate, OECD TG209). However, hydrolysis and biodegradation may release toxic byproducts over longer time scales.

Key value for chemical safety assessment

Additional information

The toxicity of MTDID 15670 to activated sludge was assessed according to the OECD 209 guideline. The test substance was melted, applied to watch glasses, and added directly to test vessels owing to difficulty of working with aqueous solutions. Some effect on nitrification was noted in a preliminary test, however overall effects were small. In the definitive test, 50% effect was not attained at 1000 mg/L. The NOEC was 100 mg/L, and the calculated EC10 was 139 mg/L. MTDID has a hydrolytic half-life too short to be determined at pH 7 and 23 °C. In an OECD 301B test, it could not be ascertained whether MTDID 15670 was inhibitory to degradation, and no meaningful degradation was observed. The expected pathways obtained by QSAR modeling of the hydrolysis product dodecyl ethyl sulfide include mercaptan (thiol) formation. The free sulfhydril groups are known to be toxic to some microorganisms.